Recently hydrolysis of phosphate esters has received extensive investigation as reported in the art due to the relevance of this chemistry to biological systems, and specifically transition metal complexes have been examined as phosphate ester hydrolysis catalysts in order to model the reactions catalyzed by the ATPase and phosphatase classes of enzymes. Such reported studies have generally employed activated p-nitrophenyl phosphate esters or phosphate anhydrides (ATP) as substrates (R. D. Cornelius, Inorg. Chem. 1980, 19, 1286-1290; P. R. Norman et al, J. Am. Chem. Soc. 1982, 104, 2356-2361 and F. Tafesse et al, Inorg. Chem. 1985, 24, 2593-2594). It has been reported that tetramine complexes of Co(III) are capable of promoting the hydrolysis of adenosine 3',5'-monophosphate (cAMP) (J. Chin et al, Can. J. Chem. 1987, 65, 1882-1884) and adenosine monophosphate (AMP) (J. Chin et al, J. Am. Chem. Soc. 1989, 111, 4103-4105). Also, it is known that many divalent cations are capable of catalyzing the hydrolysis of RNA (J. J. Butzow et al, Biochemistry 1971, 10, 2016-2027 and J. J. Butzow et al, Nature 1975, 254, 358-359). Additionally, zinc ion in the presence of imidazole buffers has been shown to catalyze the hydrolysis of the RNA dimer 3',5'-UpU at 80.degree. C. (R. Breslow et al, Proc. Natl. Acad. Sci. 1989, 86, 1746-1750).
C. A. Stein et al, Cancer Research May, 1988, 48, 2659-2668 gives a detailed review on the application of antisense oligodeoxynucleotides as modulators of gene expression and concludes by proposing a more subtle and effective approach would be to attach a chemical group to the oligomer that can result in localized catalytic hydrolysis of RNA. This technique would be more specific than the use of a radical-producing group such as iron EDTA. Stein et al theorizes that a suitable RNA hydrolysis group would be an imidazole group, which is known to be involved in phosphodiester hydrolysis in the active site of ribonuclease enzymes.
University Patents, Inc. in PCT International Patent Application published under number WO 88/04300 on Jun. 16, 1988 discloses RNA enzymes or ribozymes, acting as endoribonucleases, as catalyzing the cleavage of RNA molecules with a sequence specificity of cleavage greater than that of known ribonucleases and approaching that of the DNA restriction endonucleases, thus serving as RNA sequence-specific endoribonucleases. Ribozymes are entirely or partly comprised of RNA itself, and therefore are chemically and enzymatically highly unstable relative to Applicants' DNA-based compounds. Such instability detracts from the practical applicability of RNA hydrolysis agents.
C. B. Chen et al, J. Am. Chem. Soc. 1988, 110, 6570-6572 describes that 1,10-phenanthroline-copper(II) is effective for targeted cleavage of both RNA and DNA and thus is useful for sequence-specific cleavage of RNA. This teaching is directed to oxidative cleavage of RNA by metal complexes linked to DNA at a temperature of 65.degree. C. as opposed to the hydrolytic cleavage of RNA under physiologically relevant conditions required by Applicants' invention. The ancillary reagents, in the quantities required to drive the Chen et al oxidative degradation of RNA, are not compatible with living cells; furthermore, the 1,10-phenanthrolinecopper-oligodeoxynucleotide conjugate employed is itself degraded oxidatively under the conditions of oxidative RNA cleavage (the rate of oxidative cleavage by the 1,10-phenanthrolinecopper system is similar for both RNA and DNA).
P. G. Schultz and coworkers in a series of articles (D. R. Corey et al, J. Am. Chem. Soc. 1988, 110, 1614-1615; R. Zuckerman et al, J. Am. Chem. Soc. 1988, 110, 6592-6594 and R. Zuckerman et al, Proc. Natl. Acad. Sci. USA 1989, 86, 1766-1770) have described the preparation of site-selective DNA and RNA hydrolysis agents comprised of an enzyme (staphylococcal nuclease, ribonuclease S, or mutants of these parent enzymes) covalently linked to oligodeoxynucleotides. In one report (D. R. Corey et al, Biochemistry 1989, 28, 8277-8286), the location of the linker arm and its length were varied, which resulted in changes in catalytic efficiency and site of cleavage.
Considerable art has been developed on cleavage of RNA utilizing enzymes and ribozymes. At present the art is void of a teaching using metal complexes which cleave RNA hydrolytically at a physiologically relevant pH and temperature as synthetic analogs for enzymes or ribozymes to obtain sequence-directed hydrolysis of RNA. Sequence-directed RNA hydrolysis is highly desirable today in order to prepare catalytic antisense oligodeoxynucleotides useful as a means for inhibiting the expression of specific genes. Such RNA hydrolysis is necessary to provide a basis for catalytic antisense drug development.